Abstract

2453

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of proliferation and progression in human tumors. In addition, it has recently been identified as a transcription factor for genes involved in G1/S progression. To date five EGFR inhibitors have gained FDA approval in oncology and have demonstrated major clinical tumor regression in approximately 10-20% of advanced cancer patients. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. The underlying mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain largely unexplored. To investigate molecular mechanisms of acquired-resistance to cetuximab we established a series of cetuximab-resistant clones by chronically exposing the non-small cell lung cancer (NSCLC) cell line, H226, to cetuximab. We previously reported that cetuximab-resistant clones had a dramatic increase in the steady-state expression of EGFR due to alterations in trafficking and endocytosis. This resulted in constitutive activation of MAPK, HER3 and downstream survival signals mediated by AKT. Only MAPK signaling was blocked when resistant cells were treated with cetuximab, but this had no effect on overall growth indicating other proliferative signals play a key role in resistant cells. In this communication we report that NSCLC cancer cells with acquired-resistance to cetuximab had 1) markedly higher levels of Src Family Kinase (SFK) activity and 2) robust nuclear EGFR expression relative to parental controls. Inhibition of SFK activity using PP1, PP2 or dasatinib resulted in decreased tyrosine phosphorylation of the EGFR which resulted in decreased activity of HER3, AKT and cell survival. In addition, inhibition of SFK activity led to the loss of nuclear expression of EGFR with a subsequent decrease in Cyclin D1 and B-Myb expression. Together these data suggest that cells with acquired-resistance to cetuximab have two major proliferative pathways mediated by the EGFR: 1) membranous EGFR activation of MAPK signaling and 2) nuclear EGFR regulation of Cyclin D1 and B-Myb expression. Treatment of cetuximab-resistant cells with the combination of dasatinib and cetuximab led to profound anti-proliferative potential greater than either agent alone. This is most likely the result of inhibition of membranous EGFR mediated MAPK signaling by cetuximab and inhibition of nuclear EGFR mediated Cyclin D1 and B-Myb expression by dasatinib leading to abrogation of both proliferative compartments. Collectively, these data suggest that SFKs play a critical role in acquired-resistance to cetuximab and suggest a rationale for designing clinical trials to examine the value of treating patients that manifest cetuximab-resistance with inhibitors of SFKs in combination with cetuximab.